1. Field of the Invention
The field of this invention is assays for detecting ADP presence and formation.
2. Related Art
ATP is ubiquitous as the major power source for live organisms. ATP is used with enzymes referred to as ATPases, such as with kinases for phosphorylating proteins, for glycogenesis, and for metabolizing sugars and fats, to name only a few. In trying to discern cellular pathways and the effect of changes in the environment on these pathways, the ability to measure the amount of ADP is advantageous. Also, one may be interested in the amount of ATP in a sample, particularly where the ATP is to be monitored by the formation of ADP.
One approach for measuring ADP is to use an enzyme-coupled reaction in order to produce a readily detectable signal. Since, in many of the contexts in which one wishes to measure ADP, the amount of ADP will be very small or one will be interested in small differences in the amount of ADP between determinations, sensitive detection becomes an important factor.
In the literature, there has been a description of an enzyme-coupled reaction to determine ADP. In this method, pyruvate kinase and phosphoenolpyruvate are combined in the assay mixture and react with ADP to form ATP and pyruvic acid. Also included are pyruvate oxidase and its cofactors FAD and TPP, which transform the pyruvate to acetyl phosphate and hydrogen peroxide. The hydrogen peroxide is then detected by catalyzing its reaction with the fluorescent dye, Amplex® Red (a fluorescent dye) using horseradish peroxidase.
While the method would appear to be highly sensitive, it suffers from the problems of contamination of the various materials used in the process. Phosphoenolpyruvate spontaneously hydrolyzes to give pyruvic acid, which is involved as an intermediate in the production of hydrogen peroxide. Any adventitious pyruvic acid will result in an augmented determination, indicating a larger amount of ADP than was present. Also, buffers and assay components may be contaminated with hydrogen peroxide. The substrates one employs as the detectable species and other components of the assay may also produce hydrogen peroxide. The method therefore suffers from the potential for numerous introductions of intermediates that will alter the result and provide for erroneous determinations. In addition, it is well known that ATP will spontaneously hydrolyze to ADP and phosphate, so that the ATP may be contaminated to varying degrees with ADP depending upon the manner in which the ATP is prepared, stored and handled. Since adventitious presence of ADP in the assay mixture will substantially compromise the assay, it is important to minimize the amount of ADP present from other than the reaction of interest.
In developing an assay for commercialization, there are a number of considerations that guide the protocol employed. Desirably, the maximum number of agents employed in the assay is combined as a single reagent, so as to minimize the number of measurements and additions that must be performed. The greater the number of measurements and additions that must be performed, the greater the errors that are likely to be produced. Particularly, where an assay requires a large number of reagents, it is especially desirable to be able to combine them in large amounts and mix the reagents homogeneously, so that they are accurately measured in the proper proportions and can dissolve and rapidly interact during the assay.
Because of the importance of the determination of ADP, providing for an accurate, reasonably rapid method of measurement, where one can use the protocol in both medium and high throughput screening, is very desirable.
Relevant Literature
An abstract presented at the SBS 2002 meeting on Sep. 22-26, 2002, by Juan-Manuel Dominguez, et al., in the section Novel Detection Technologies and Assay Formats, ID:2322, states: “A new fluorogenic method for the detection of ADP is described. It is based on the sequential action of pyruvate kinase and pyruvate oxidase to generate hydrogen peroxide, which is utilized by peroxidase to convert non-fluorescent Amplex® Red (a fluorescent dye) into fluorescent resorufin. The method is amenable to on-line kinetics and has proved to be very sensitive, capable of detecting 1 micromolar ADP, and thus suitable to be used to measure kinase activities. To support this idea, bacterial thymidylate kinase has been screened using this method to monitor enzyme activity against a collection of 500,000 compounds. Z-prime values were consistently maintained around 0.80 throughout the whole campaign. As a result, potent inhibitors of the enzyme have been found, yielding IC50 values in the nanomolar range.”
Also of interest are Finer et al., U.S. Pat. No. 6,410,254, Eiji, et al., “Pyruvate oxidase, its preparation and use,” EPA 0,274,425 and Kiianitsa, et al., “NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities,” 2003 Analytical Biochemistry 321:266-271. Japanese Application Publication Number 61-092598, or Application Number 59-214386, “Method of Determining ADP,” describes an ADP assay using pyruvate kinase and pyruvate oxidase.